ITMI20002476A1 - ADDINTS OF QUINOLONIC ANTIBACTERIALS WITH NATURAL POLYSACCHARIDIC POLYMERS - Google Patents

Description

Description of the industrial invention entitled:

'Adducts of quinolone antibacterials with natural polysaccharide polymers.

FIELD OF THE INVENTION

The present invention relates to adducts of natural polysaccharide polymers with active principles belonging to the class of quinoionic drugs with improved therapeutic efficacy.

STATE OF THE TECHNIQUE

Many of the problems that arise during treatment with chemotherapeutic agents derive from the fact that the active principles often do not possess the optimal pharmacological and pharmacokinetic characteristics to carry out their antibacterial action. An approach used in the pharmaceutical field to overcome drawbacks due to poor bioavailability or limited duration of action is to bind the antibiotic molecule to natural or synthetic, inert and biocompatible polymers. The formation of complexes or conjugates with a covalent or ionic bond is sometimes advantageous. Takeda patent EP 0392487 describes an ionic bond complex between natural sulfonated polymers and an anthracycline which is thus more stable at neutral pH.

In a Shionogi patent (EP 0438747) a natural soluble polysaccharide is added to a glycopeptide antibiotic, with the function of support during the lyophilization of the finished product.

In patent EP0428486, Sandoz prolongs the half-life of Polymyxin B by binding it to a natural or synthetic polymer with a covalent bond.

The polymer conjugated with the antibiotic can be active as such or, if the "linker" is biodegradable, it behaves like a prodrug which is subsequently bioactivated in vivo, releasing the antibiotic. However, this approach is expensive and complicated since it is necessary to chemically characterize the compound, as it appears to be a newly established product, and then re-study its pharmacotoxicology, pharmacokinetics and clinical efficacy.

SUMMARY OF THE INVENTION

The Applicant has now surprisingly found adducts of quinolone antibacterials, characterized by the fact that the percentage of said active ingredients is between 40 and 80% by weight with respect to the total weight of the adduct.

The adducts according to the present invention are in the form of powder or in the form of an aqueous solution which preferably contains them in a concentration ranging from 0.1 to 1% by weight calculated on the total weight of the aqueous solution.

A further object of the present invention is the process for preparing the above adducts which in particular comprises the following steps:

a) the water solution of the polysaccharide polymer is prepared in a percentage between 20 and 60% by weight of the total weight of the adduct to which the active principle is subsequently added, so that the concentration of the adduct in the final solution aqueous solution is preferably included in the aforementioned ranges, b) the aqueous solution thus obtained is filtered, if the adduct is to be prepared in the form of an aqueous solution,

c) the water is removed and the adduct according to the present invention is obtained in powder form.

A further object of the present invention are pharmaceutical compositions containing the adducts object of the present invention in combination with suitable excipients and / or diluents.

DETAILED DESCRIPTION OF THE INVENTION

The object of this invention represents an optimal form of oral and parenteral administration of kinoionic antibacterial drugs for the treatment of infections caused by Gram-negative and Gram-positive bacteria.

More specifically, the present invention relates to adducts between an active principle, belonging to the class of Quinoions, and a natural biocompatible polysaccharide, between which an interaction based on non-covalent and non-ionic bonds is established.

The adducts can be administered both orally and parenterally, according to the chemical-physical characteristics and the pharmaco-dynamic and pharmaco-kinetic profile of the active ingredient.

The active ingredients used in the adducts according to the present invention are preferably nalidixic acid, pipemidic acid, cinoxacin, norfloxacin, ciprofloxacin, pefloxacin, enoxacin, ofloxacin and its levo form levofloxacin. They are mainly used in the treatment of infections caused by Gram-negative and positive bacteria and their mechanism of action consists in blocking DNA synthesis by inhibiting the DNA gyrase enzyme.

In more detail, norfloxacin is effective in urinary, enteric and gonorrhea infections at a dosage of 800-1200 mg / day via oral intake.

Ciprofloxacin is indicated for urinary, respiratory, gastrointestinal, skin infections and osteomyelitis, as it is effective against Gram-negative organisms (E. Coli, Klebsiella, Salmonella typhi, Neisseria gonorrhoea et meningitidis, Haemophilus influenzae), Gram-positive (Staph . aureus et epidermidis, listeria) and Mycobacterium tubercolosis. It can be administered orally as hydrochloride with daily doses of 500-1000 mg or in the most severe cases intravenously at the rate of 800-1200 mg / day as a side. (A. Kucers et N. Mck. Bennett, The use of Antibiotics, 4th ed., Heinemann Medicai Books, 1989).

According to a particularly preferred solution of the present invention, the percentage of the quinoionic antibacterial with respect to the total addote is 60%.

Dextrans, inulin and, for oral preparations, pharmaceutical grade maltodextrins have been preferred among the natural polymers then isacca ridici.

Dextrans are polymers made up of linear chains of α-D-glucose molecules and can have very different molecular weights, in a range from 1000 Dalton (dextran 1) to 110000 Dalton (dextran 110) and do not undergo enzymatic degradation. The hydrophilicity and therefore the solubility in water decreases with the increase of the molecular weight. Dextrans at molecular weights below 4000 are completely excreted in the urine within 48 hours, while at higher molecular weights they remain in circulation for longer periods of time. Preferred in the present invention are dextrans 4 to 70.

Maltodextrins consist of a branched polymer of maltose and dextrins in which the D-glucose units are mainly linked by bond to (1-4), but in the branched tracts also to (1-6). They have molecular weights ranging between 900 and 9000 Dalton.

Inulin is also a natural polysaccharide with a linear structure consisting of fructose molecules with a molecular weight of 5000, it does not undergo enzymatic degradation and is excreted in the urine, so much so that in the medical field it is used as a diagnostic means of renal function.

The selected natural polysaccharides are biocompatible and inert and therefore their adducts with the quinoions do not present toxicological and immunological problems.

The preparation of the adducts involves the solution of the polymer in water, in a percentage that can vary from 60% to 20% by weight, and the subsequent addition of the active principle. This makes possible the hydrophilic interaction of carboxylic and amino groups of the antibacterial with those of the polymer. Thus, weak hydrogen-like bonds are established with lower interaction forces than covalent or ionic bonds (Remington's Pharmaceutical Science, 18th ed., P. 186).

The polymer and the drug are dissolved in distilled water (with possible buffers and preservatives) and a filtration is carried out to obtain a clear solution. Instead, to obtain the adduct in solid form, the solvent is removed from the adduct through a freeze-drying or nebulization process (spray dryer), obtaining the adduct in solid form.

For the preparation of injectable forms, distilled, depyrogenated and sterile water (p.p.i.) is used and the solution is filtered, with filters with porosity from 0.1 to 0.2 μm, then divided into sterile vials, depyrogenated and sterilized, and preferably freeze-dried. The solution can be readily reconstituted by adding an aqueous solvent such as water p.p.i. or physiological solution.

Distilled water is used for the preparation of oral forms and the solution is filtered through 0.45 μm porosity filters. The solvent is preferably removed by nebulization, as a solid and porous adduct is obtained, suitable for the preparation of pharmaceutical forms for oral use, such as tablets, capsules and granulates.

The adducts obtained were subjected to determination of the titer in active principle by HPLC using a Perkin-Elmer LC75 chromatograph with LC Pump 414-T Karitron Analytical injector, Waters integrator recorder and a Lichrosorb RP18 column.

The in vitro microbiological activity of these adducts compared to that of the corresponding quinoions was determined both by measuring the inhibition halos developed in the plate, and by determining the minimum concentrations capable of inhibiting bacterial growth in the test tube. The in vivo activity was evaluated on mice previously infected with a strain sensitive to the quinoions under examination.

These adducts favorably modify the pharmacokinetic and pharmacological characteristics of the antibacterial. In fact, an adduct with low molecular weight polysaccharide can increase the solubility of a poorly soluble antibacterial and consequently, if administered orally, its bioavailability. In case of parenteral administration an adduct with high molecular weight polysaccharide can prolong the residence time of the drug in circulation and therefore its effect.

Furthermore, the presence of reduced doses of the active principle reduces the toxicity with the same antibacterial activity.

The adducts in the form of aqueous solutions object of the present invention can be prepared with a process which in particular comprises the following steps:

a ') the active ingredient is dissolved in distilled water,

b ') the aqueous solution obtained in the previous stage is filtered,

c ') the water is removed preferably by lyophilization from the product obtained in the previous stage,

d ') the aqueous solution is reconstituted by adding water or other solvent p.p.i. in which the polysaccharide polymer is dissolved, so that the concentration of the adduct in said aqueous solution is preferably between 0.1 and 1% by weight.

The adducts of the present invention have been obtained with simple, fast and economical processes if compared to traditional synthetic approaches, which provide for the formation of a covalent or saline bond between the antibacterial and the polymer, and therefore are also less expensive. Some examples of preparation of the adducts reported below illustrate the invention without however limiting it.

EXAMPLE 1 - Preparation of the solid adduct maltodextrin 40% -norfloxacin 60%

Under magnetic stirring, 0.67 g of maltodextrin M500 are dissolved in distilled water. 1 g of norfloxacin is added to the solution by buffering with 1N HCl up to pH 4.8 and a final volume of 1 L is made with distilled water. The solution, after filtration on a filter with a porosity of 0.45 μm, is subjected to nebulization with a mini spray dryer (Mini Buchi). The jet pressure is 800 mbar, the inlet temperature of 130 ° C and the outlet temperature of 50 ° C and the suction is kept at maximum capacity.

1.51 g of adduct are obtained in the form of an ivory-colored granular powder, soluble in water and with a norfloxacin content of 59.5% by weight. With the addition of appropriate excipients, the adduct is suitable for being formulated as capsules, sachets, and after compression, tablets.

EXAMPLE 2 - Preparation of the solid adduct dextran 50% -norfloxacin 50%

1 g of dextran 5 is dissolved in 1 L of distilled water and 1 g of norfloxacin is added to the solution, bringing the pH to 4.8 by adding 1N HCl. It is filtered with a 0.45 μm porosity filter. and the solvent is removed by nebulization as described in example 1. 1.87 g of adduct are obtained as a light whitish powder, with a norfloxacin content of 49% by weight. The powder with the addition of suitable excipients can be divided into sachets or formulated in capsules or tablets directly.

EXAMPLE 3 - Preparation of the solid adduct maltodextrin 50% ciprofloxacin 50%

In 1 L of distilled water 2 g of maltodextrin M500 are solubilized under magnetic stirring. 2.5 g of ciprofloxacin lactate are added (equal to 2 g of active principle), leaving it under stirring and adding HCl 1N q.s. until total solubilization. The solution is filtered and sprayed following the same parameters as in the previous example.

4.2 g of adduct are obtained as a light ivory-colored powder, with a ciprofloxacin content of 49.7% by weight. The powder with the addition of suitable excipients can be divided into sachets or formulated in capsules or tablets directly.

EXAMPLE 4 - Preparation of the adduct inulin 40% -ciprofloxacin 60% Under magnetic stirring in 800 ml of water p.p.i. 1.6 g of Inulin are dissolved and 3 g of ciprofloxacin lactate (ciprofloxacin 2.4 g) are added adding q.s. of 1N HCl for complete solubilization. Further water is added p.p.i. to correct the total volume to 1 L. The solution is filtered with a 0.2 μm porosity sterilizing filter and poured into a bulk freeze-drying tray. The freeze dryer is programmed to run the freeze drying cycle at the following temperatures: -35 ° C for pre-freezing, -10 ° C during freeze drying, 10 ° C for primary drying and 35 ° C for secondary drying, maintaining the vacuum on values of 2.210 <-2> mbar. 4.53 g of adduct are obtained in the form of a compact lyophilisate, soluble with the addition of physiological solution, and with a ciprofloxacin title of 58.3% by weight. The bulk adduct is finally divided into containers by operating in a sterile environment under a laminar flow hood. EXAMPLE 5- Preparation of the adduct dextran solution 40% -ciprofloxacin 60%

In a volume of 800 ml of water p.p.i. 1.6 g of dextran 5 are dissolved under magnetic stirring, 3.1 g of ciprofloxacin lactate equal to 2.43 g of ciprofloxacin are added, correcting the pH with 1 N HCl until complete solution. The solution is brought to 1.5 L with p.p.i.water, filtered with a sterilizing filter of 0.2 μm porosity and divided into depyrogenated and sterilized vials, at a rate of 37.5 ml / vial. (The title of ciprofloxacin in the solution is 60.5%.

EXAMPLE 6 - Preparation of the solid adduct dextran 40% -ciprofloxacin 60%

The solutions of the adduct of example 5 are subjected to lyophilization following the same cycle described in example 4. The lyophilisate obtained is compact with a ciprofloxacin title of 60.4% by weight. It can be readily reconstituted for a slow infusion with the addition of 30 ml of physiological solution.

EXAMPLE 7- Preparation of the adduct in inulin solution 40% -ciprofloxacin 60%

In a volume of 500 ml of water p.p.i. 2 g of ciprofloxacin lactate equal to 1.57 g of ciprofloxacin are dissolved under magnetic stirring, correcting the pH with 1N HCl until complete solution. The solution is brought to 520 ml with p.p.i water, filtered with a sterilizing filter of 0.2 μm porosity and divided into depyrogenated and sterilized vials, at a rate of 20 ml / vial. The vials are placed in a freeze dryer programmed to perform the cycle described in example 4. The lyophilisate obtained is compact, with a ciprofloxacin title of 60.3%. Dissolving the contents of each vial with 15 ml of a solvent based on physiological solution and 40 mg of inulin constitutes the extemporaneous adduct solution.

EXAMPLE 8 - Determination of the halos of inhibition

Regarding the in vitro antimicrobial activity tests of norfloxacin and ciprofloxacin, the inhibition halos were obtained by the diffusion method (FUI X ed., P. 135), using a Mueller Hinton Medium Difco culture medium (low thymine and thymidine content) conforming to NCCLS MG-P standards in sterile 9 cm diameter Petri dishes and Tryptic soy broth (Difco).

For the sensitivity test, both filter paper discs soaked in quinoionic antibacterial in determined concentrations, and the corresponding adduct discs, in which the total dose is the same, but the weight content of the active ingredient is lower, were prepared.

To produce bacterial growth, 1 strain of Stafilococcus aureus ATCC 6538 P (strain A) and 2 strains of field Stafilococcus aureus (strain B and C) were used. Brodocultures were set up by placing a bacterial patina loop of a 12 h agar culture in a tube containing 5 ml of Tryptic Soy Broth Sterile and then incubated at 37 ° C for 4 h.

If necessary, the suspensions were diluted with sterile water to obtain a turbidity similar to the reference standard (0.5 Me Farland). 150 milliliters of Mueller Hinton Medium were sterilized in an autoclave at 121 ° C for 15 ', part was placed in sterile 9 cm diameter Petri dishes and allowed to solidify, part was cooled to 45 ° C s and inoculated with 1 , 5 ml of the bacterial suspensions previously prepared and poured on the surface of the solidified agar at the rate of 10 ml per plate.

The seeded plates were kept to dry open under a laminar flow hood for 15 'and, after this time, the discs were deposited on the agar surface with fine-tipped tweezers that were flame-sterilized and cooled. In each plate 4 disks were placed: the adduct and the kinoionic antibacterial in the two concentrations that we wanted to compare. The plates thus prepared, 5 for each test, were incubated upside down at 37 ° C for 16 h and after this time the inhibition zones were determined.

The results obtained are reported in Table I.

Table I.

EXAMPLE 9 - Determination of the minimum inhibitory concentration (MICI in vitro

The MIC values were determined by the method of dilutions in liquid medium (F. Pasquinelli, Manual for laboratory technicians, vol. Il, p. 1286-89, 1968), using a Mueller-Hinton broth at pH 7.4, sterilized at 120 ° C for 20 minutes. In a series of tubes containing a fixed volume of medium, scalar concentrations by doubling of the active principle were added, from 0.1 μg / ml to 10 μg / ml. The same procedure was also followed for abductees.

All tubes were added with a drop of 1:10 dilution of the broth culture and incubated at 37 ° C for 18-24 hours. After this time, the tube with the highest dilution in which no turbidity was observed was considered to contain the lowest inhibitory concentration.

The results obtained are reported in Table II.

Table II

EXAMPLE 10 - In vivo activities

Solutions at various dilutions with purified water for oral use of the two antibacterials and respective adducts were administered to common albino mice weighing 28 g. each every 24 hours for five days. They had previously been infected with Staphylococcus aureus (which did not show resistance to the antibacterials under consideration), and subsequently developed ulcerative dermatitis with localized abscesses.

After the treatment, a bacteriological control was carried out for re-isolation, by carrying out swabs on the skin of the animals and proceeding with the inoculation of the material taken in a plate. The results are reported in Tables III and IV.

Table III

Table IV

The adducts of the present invention between quinoionic antibacterials and natural polysaccharide polymers have shown, in the in vitro and in vivo antimicrobial activity tests in mice, an activity comparable or slightly higher than that of the active principle alone, although containing it in lower quantities, thus reducing the cost of therapy.

This translates to equal efficacy in a reduction of adduct toxicity.

Furthermore, these adducts are obtained with a fast, economical and suitable process for the subsequent preparation of parenteral forms and, with suitable excipients, of oral forms for the treatment of infections due to Gram-positive and Gram-negative bacteria in animals and in man.

Claims (18)

CLAIMS 1. Adduct of a quinoionic type antibacterial with a polysaccharide polymer of natural origin, characterized in that the percentage of said active principle in the adduct is between 40 and 80% by weight of the total weight of the adduct. 2. Adduct according to claim 1 characterized in that it is in the form of an aqueous solution. 3. Adduct according to claim 2 characterized in that it is contained in said aqueous solution in concentrations ranging from 0.1 to 1% by weight of the total weight of said aqueous solution. 4. Adduct according to claim 3, characterized in that it is in the form of a powder. _ 5. Adduct according to claim 4 characterized in that it is obtained by nebulization or lyophilization of the aqueous solution according to claims 2 and 3. 6. Adduct according to any one of claims 1-5, characterized in that the content of quinoionic type antibacterial is equal to 60% by weight of the total weight of the adduct. 7. Adduct according to any one of claims 1-6, characterized in that the quinoionic antibacterial is selected from the group consisting of nalidixic acid, pipemidic acid, cinoxacin, norfloxacin, ciprofloxacin, pefloxacin, enoxacin, ofloxacin, levofloxacin. 8. Adduct according to any one of claims 1-7, characterized in that said polysaccharide polymers of the natural type are selected from the group consisting of: dextrans, inulin, pharmaceutical grade maltodextrins. 9. Adduct according to claim 8, characterized using dextrans from 4 to 70. 10. Process for preparing the adduct according to any one of claims 1-9, comprising the following stages: a) the water solution of the polysaccharide polymer is prepared in a percentage between 20 and 60% by weight of the total weight of the adduct to which the active ingredient is subsequently added, b) the aqueous solution thus obtained is filtered, if the adduct is to be prepared in the form of an aqueous solution, according to claims 2 and 3; c) the water is removed and the adduct is obtained in the form of a powder according to claim 4. The process according to claim 12 for preparing the adduct in the form of an aqueous solution according to claim 3, characterized in that in step (a) the concentration of the adduct in said aqueous solution is between 0.1 and 1 % by weight. The process according to claim 10, characterized in that for the preparation of the adduct in the form of a sterile aqueous solution for injectable use in step (a) distilled, depyrogenated and sterile water (p.p.i.) is used, and in step (b ) the solution is filtered with filters having porosity from 0.1 to 0.2 μm, and step (c) is carried out by lyophilization. 13. The process according to claim 10, characterized in that for the preparation of the adduct to be used for oral use in the. step (a) distilled water is used, in step (b) the solution is filtered on filters with porosity 0.45 μm, and in step (b) the solvent is removed by nebulization. 14. Process for preparing the adduct in the form of an aqueous solution according to claim 2 or 3, comprising the following stages: a ') the active ingredient is dissolved in distilled water, b ') the aqueous solution obtained in the previous stage is filtered, c ') the water is removed preferably by lyophilization from the product obtained in the previous step, d ') the aqueous solution or other solvent p.p.i. is reconstituted. by adding water in which the polysaccharide polymer is dissolved. 15. The process according to claim 16, characterized in that the concentration of the adduct in the aqueous solution obtained in step (d ') is between 0.1 and 1% by weight. 16. Pharmaceutical composition containing as active principle at least one adduct according to any one of claims 1-9, in combination with suitable excipients and / or diluents. 17. Composition according to claim 15, for oral use in the form of tablets, capsules, granulates. 18. Pharmaceutical composition according to claim 15 for injectable use in the form of an aqueous solution that can be prepared at the moment of use by adding water p.p.i. to the adducts prepared with the process according to claim 11.